RNA is an important target for the design of small molecule therapeutics and probes of function. It is an underutilized target, however, because of the limited information available about how RNA motifs interact with small molecules. This proposal describes studies to identify RNA motif-small molecule partners and investigate important features in both the RNA and small molecule that govern molecular recognition. The RNA motifs used are small internal and hairpin loops that are commonly found in biologically important RNAs such as pre- microRNAs, underexploited and important RNA drug targets with secondary structures similar to our RNA motifs and no known tertiary structure. Two synergistic methods are used to enable parallel probing of features in the RNA and the small molecule. The first method, 2D Combinatorial Screening (2DCS), screens two libraries simultaneously (an array-immobilized small molecule library and an RNA library) to identify RNA motif-small molecule partners. RNAs are harvested directly off the array from ligand-functionalized positions, cloned, and sequenced. Selected RNAs and RNA motif-small molecule complexes can be studied by NMR and optical melting experiments to interrogate their structures, flexibilities, and thermodynamic stabilities. The second method, Structure Activity Relationships Through Sequencing (StARTS), assigns relative binding affinities from the output of 2DCS via the occurrence of RNA loops in sequencing data. Multiple selected sequences are ligated together and cloned, yielding more sequence data per single sequencing reaction. If information about RNA-small molecule interactions were available, an RNA target's secondary structure could be searched for one or several motifs to which a small molecule partner was identified, and the small molecule modules custom-linked to accommodate each site in the target RNA. This may eliminate the need to subject each new RNA target to a high throughput screening assay. The specific aims for this study are: 1.) Characterize the molecular recognition of 6'-N-5-hexynoate kanamycin A by internal loops with AC pairs, the consensus loop determined from a previously completely selection. AC loops are present as mutations in tRNAs that cause disease. 2.) Identify and study the RNA internal loops selected to bind members of an aminoglycoside library. 3.) Use StARTS and 2DCS to streamline identification and statistical analysis of the internal loops that bind 6'-N-5-hexynoate neamine. 4.) Expand the results in Aims 2 and 3 to identify RNA hairpin loop-aminoglycoside interactions. 5.) Identify interactions between a peptoid library and RNA internal loops and hairpin loops via StARTS and 2DCS. PROJECT NARRATIVE RNA is an important biomolecule that is associated with diseased states, however, very few drugs elicit their effects by interacting with RNA. The goal of the proposed research is to understand how drug-like molecules interact with RNA to design therapeutics or probes of RNA function.